The locations of the majority of post-transcriptional modifications in rRNA are near the functional site of the ribosome. However, the identification of these modifications remains problematic and their function remains unknown. The long term goal of this research is the characterize and understanding the function of these modifications, especially concerning their role in protein synthesis, is the development of improved methods for their structural characterization. Specifically, a mass spectroscopy-based protocol will be used to determine the sequence location and identity of post-transcriptionally modified nucleosides in rRNA. Selective cleavage of rRNA with endo- and exonucleases followed by mass spectrometric analysis of these digestion products will permit the identity and sequence location of post- transcriptional modifications to be determined. This determination is made by comparing experimentally measured mass values to that predicted from the gene sequence. Sites of modification (except for pseudouridine) are manifested as an increase in the measured molecular weight. The identification of pseudouridine, the only silent modification, will be performed using specific chemical reactions that shift the mass of all pseudouridine residues permitting their identification using mass spectrometry. The initial development of these methods will be performed with Escherichia coli 16S and 23S as the model systems. The methods developed here are applicable to a wide range of nucleic acids (e.g., snRNA, mRNA, tRNA) which contain modified nucleosides. This research will provide a rapid and accurate method for the sequence analysis of rRNA and will permit more detailed studies on the functional importance of post-transcriptional modifications in protein synthesis.